In the last few decades, substantial and continuous works within the field of sialobiology have shed light on the importance of sialic acids in the biology of both eukaryotes and prokaryotes. In eukaryotes, sialic acids are involved in the regulation of important biological functions, but they are also involved in interactions with bacteria and viruses. A few pathogenic bacteria decorate themselves with sialic acids on their surfaces to avoid the host immune system, whereas many viruses use these sugars as receptors to enter the host.
Sialic acids are sugars found on the surface of both prokaryotic- and eukaryotic cells and belong to the family of nine carbon α-keto acidic monosaccharides. N-acetylneuraminic acid (Neu5Ac) is the most frequent sugar among this family. Extensive research has been done after the discovery of the Neu5Ac in 1936 due to its interesting and important biological roles. The application of sialic acid and its derivatives are growing; they are used in diagnostic research because an elevated concentration of free serum sialic acid is indicative of several diseases, the concept of sialic acid as a glyconutrient is emerging due to the fact that it is important in fetal brain development and analogues of sialic acids are considered potential antiviral agents. One of the most successful examples of the latter is Zanamivir. Consisting of a simple modification of Neu5Ac it inhibits neuraminidases of both influenza virus A and B, and it is also used commercially as protection against the highly virulent H5N1 strain (birdflu).
Many promising therapeutic applications of Neu5Ac have led to an increased interest in developing efficient methods for its production. Isolation of Neu5Ac from natural sources such as egg yolk, edible bird's nest and milk is hampered by low yields, hence, inappropriate for large scale production. Neu5Ac can be produced by de novo chemical synthesis. However, the structural features of the molecule render inherent challenges concerning the correct stereospecificity. Chemoenzymatic synthesis has been reported, but the subsequent steps of chemical addition can be cumbersome. Therefore, highly stereospecific and simple enzymatic methods are economically, and perhaps also environmentally better alternatives for large scale production of Neu5Ac, since the enzyme catalyzed reaction result in the formation of stereo- and regiochemically defined products with significant rate acceleration (Koeller and Wong, 2001).
Sialic acid synthase and N-acetylneuraminate lyase (NAL, or sialic acid aldolase), are enzymes which can produce N-acetylneuraminic acid by the condensation of N-acetylmannosamine (ManNAc) with phosphoenolpyruvate (PEP) (Warren and Felsenfeld, 1962) or pyruvate, respectively.
Sialic acid synthase has been recombinantly produced from different organisms, among others Aliivibrio salmonicida (Gurung et al., 2013). However, there is no report of commercial availability of this enzyme for use in industrial scale. This is probably due to the high cost of the cofactor PEP. NAL, on the other hand, is an enzyme which is available commercially and is being used to produce Neu5Ac and its analogues. It is a class I lyase/aldolase which catalyzes the reversible cleavage of Neu5Ac to yield pyruvate and ManNAc, with an equilibrium favoring Neu5Ac cleavage* (Brug and Paerels, 1958; Comb and Roseman, 1958). Its biological role is to cleave Neu5Ac, however, at favorable conditions, the reverse aldol condensation reaction can be utilized in vitro to synthesize Neu5Ac and its derivatives from pyruvate and ManNAc (Auge et al., 1984). The use of NAL instead of sialic acid synthase to produce Neu5Ac commercially is favorable because of the relatively low price of pyruvate compared to PEP. In addition, NALs generally tolerate a wide range of acceptor substrates which can be useful for synthesis of Neu5Ac analogues (Machajewski and Wong, 2000).
The production of Neu5Ac through the NAL synthetic reaction is sometimes coupled with an alkaline or enzymatic epimerization of N-acetylglucosamine (GlcNAc) to N-acetylmannosamine as a first step, because GlcNAc is a significantly cheaper starting material than ManNAc and the production costs will be reduced. This chemical epimerization is performed under alkaline conditions (above pH 9). Known NALs from e.g. E. coli, Clostridium perfringens or Pasteurella multocida however are typically active at a pH ranging from 7 to 9. Under alkaline conditions, however, these NALs are not only are unstable, but also loose activity to a great extent (Blayer et al., 1999). Hence, the unfavourable stability and activity profiles seen for these enzymes under alkaline conditions provides a boundary to the operating conditions involved in an integrated alkaline epimerization and NAL-catalyzed biotransformation process.
Thus, there is a need in the art for methods which can provide neuraminic acid or derivatives thereof such as N-acetylneuraminic acid (Neu5Ac) in high yield and in a more cost-effective manner.